Cellulosic materials are an attractive source for biofuel production, given the availability of agricultural residues that do not directly compete with food sources. However, fermentation of cellulosic biomass is problematic. Stressful byproducts generated during pre-processing, coupled with the unique composition of pentose and hexose sugars, limit microbial ethanol production. Significant attention is therefore being dedicated towards engineering stress tolerance microbes for cellulosic fermentation. Saccharomyces cerevisiae has been the organism of choice for ethanol production, because of its inherent ethanol tolerance. However, high ethanol levels can still inhibit viability and fermentation, and engineering greater ethanol resistance is therefore an important step for improved bioethanol production.
Ethanol affects many cellular processes, including membrane fluidity, protein stability, and energy status. Recent genetic screens have implicated additional genes important for ethanol tolerance, including those involved in vacuolar, peroxisomal and vesicular transport, mitochondrial function, protein sorting, and aromatic amino acid metabolism. Despite the attention to the mechanism of ethanol tolerance, significant gaps in understanding this important mechanism still exist. Several studies have investigated the global gene expression response to ethanol. However, mutational analysis shows that most genes up-regulated by ethanol are not required for ethanol tolerance. Thus, gene expression responses in a single strain are poor predictors of genes important for tolerance of the initial stressor. The inventors maintain that the role of stress dependent gene expression changes is not to survive the initial stress, but rather to protect cells against impending stress in a phenomenon known as acquired stress resistance. When cells are pretreated with a mild stress, they often acquire tolerance to what would otherwise be a lethal dose of the same or other stresses. Consistently, the gene expression response triggered by a single stress treatment has no impact on surviving the initial stress, but instead is critical for the increased resistance to subsequent stress. However, it remains true that relatively few of the previously-observed expression changes are important for subsequent tolerance of a particular stress.
Furthermore, the field's understanding of the physiological and transcriptional response to ethanol has been further narrowed since most studies focus on laboratory derived strains. While ethanol tolerance and adaptation have been explored in sake, wine, and industrial yeast strains, investigators have only recently begun to appreciate the physiological diversity of natural yeast isolates. Wild yeast isolates from diverse environments have widely varying phenotypes under various conditions, and many of these phenotypes may be related to variation in gene expression.
Thus, it can be appreciated that identifying genes related to ethanol tolerance has posed a substantial challenge to the field. Accordingly, a need exists in the field to identify genes that influence ethanol tolerance in yeast, and consequently engineer recombinant strains of yeast capable of increased ethanol yields.